In the past two years a new strategy was developed for the construction of live attenuated influenza A virus vaccine strains in which attenuating mutations are introduced into a cDNA copy of the PB2 gene by site- directed mutagenesis after which the mutant PB2 gene (in the form of full-length RNA transcripts of cDNA) is rescued into an infectious virus. This year we extended these initial findings by demonstrating the feasibility of introducing a putative ts mutation into the PB2 gene at amino acid (aa) residue 112, 265, 556, or 658 and found that each mutation indeed specified the ts and attenuation (att) phenotypes. Viable mutants were then constructed that had two or three ts mutations in the PB2 gene of the A/AA/6/60 virus. The AA mutant PB2 transfectants that possessed two or three PB2 ts mutations were highly attenuated and genetically stable in rodents and yet were able to induce protection against challenge with wild type virus. Sequential addition of single ts mutations into the PB2 gene brought about a step-wise increase in both temperature sensitivity and attenuation of the resulting transfectant virus. Such a PB2 gene bearing multiple mutations could be used alone, or in conjunction with another attenuating gene, to attenuate new epidemic influenza A viruses as they emerge in nature. The PB2 gene offers a distinct advantage over viral surface glycoprotein genes, such as the hemagglutinin or neuraminidase, as a site for an attenuating mutation(s) because it occupies an internal location in the virion and hence plays a minor role if any in inducing immunity to infection. This means that the PB2 gene (or other internal genes) can be transferred from an attenuated donor virus to any new influenza A virus by gene reassortment without compromising the immunogenicity of the new virus. Formulation and validation of this new strategy ushers in a new era in the development of live attenuated vaccines for influenza A virus. For the first time defined multiple attenuating mutations have been introduced by site-directed mutagenesis into a viral gene encoding an internal protein that can be readily transferred to new epidemic variants of the virus to achieve the desired optimal balance between attenuation and immunogenicity required of a safe and effective live influenza A virus vaccine.